The maintenance of genomic integrity following DMA damage depends on the coordination of the cell cycle checkpoint controls and DNA repair. Increasing evidence suggests that the integrity of the DMA damage- signaling pathway is essential for the prevention of neoplastic transformation, since many proteins involved in this pathway are frequently mutated in human cancers. Thus, a better understanding the DNA damage-signaling pathway will help us elucidate the pathway that is critical for the maintenance of genomic integrity and for the prevention of tumorigenesis. 53BP1 (p53-binding protein 1) was first identified in a yeast two-hybrid screen as a protein that interacts with the central DMA-binding domain of p53. In the last few years, we and others have demonstrated that 53BP1 is involved early in DNA damage-signaling pathway and plays a critical role in DNA damage checkpoint control and DNA repair. Moreover, we have shown that loss of 53BP1 greatly facilitates tumorigenesis in vivo, supporting that proper DNA damage responses are essential for the maintenance of genomic stability and cancer prevention. However, the mechanisms by which 53BP1 operates and the complexity of the DNA damage responses have not yet been adequately addressed. In this proposal, we will study the multiple functional motifs in 53BP1 in Specific Aim 1. The goal here is to elucidate how 53BP1 acts as a DNA damage mediator through its interactions with various upstream and downstream protein components involved in DNA damage pathways. In Specific Aim 2, we will explore the potential redundant functions between 53BP1 and another mediator of DNA damage checkpoint MDC1. 53BP1 rapidly relocalizes to the sites of DNA breaks. Recent studies suggest that a protein methyltransferase Dot1-dependent Lys-79 methylation of histone H3 is required for this initial recruitment of 53BP1. In Specific Aim 3, we will generate Dot1 knockout mice and explore the role of Dot1 and Dot1 -dependent H3 methylation in the regulation of DNA damage responses including 53BP1 localization. Collectively, these genetic, structural and biochemical analyses of 53BP1 protein will reveal, at molecular details, how 53BP1 participates in DNA damage responses and contributes to tumor suppression. [unreadable] [unreadable] [unreadable]